OJT: How to do SDS PAGE

Tuesday, January 10, 2017

This is where we are going to use our SDS Gels.
Prepare extracted samples for 30 percent SDS page. Let’s use the tray with crashed ice, pipetor, 96-well plate, extraction buffer, sample buffer and protein extract (the extracted samples). Thaw the samples and use a vortex to mix and place the 20 microtubes with protein extract in the centrifuge and let it spin for five minutes.
Add label to the 96-well plate rice and maize containing 10 wells each. Set the pipettor to 50µl for the sample buffer. Add sample buffer per well. Set again the pipettor to 35µl for extraction buffer and add again for 20 wells. Set again the pipetor to 15µl for protein extract and add to each well where sample buffer and extract buffer were placed. Finally, heat the 96-well plate at 95ºC for five minutes and place it immediately on ice and the proteins are ready use.
Ten percent acrylamide gel with 10 wells will be use for loading. The gel will be added to SDS gel machine and pour a running buffer on top. Set the pipettor 10µl for loading of samples. The loading format was. M1, M2, M3, M4, M5, R1, R2, R3, R4, R5 and M6, M7, M8, M9, M10, R6, R7, R8, R9, R10 (it depends on your samples what labels will you use). After loading everything, the machine must be set to 160 volts for 80 minutes.
As soon as the SDS gels with proteins samples were ready, the gels with a notch must be transfer in each tub, and add gel fixer then place the tub on the shaker for 30 minutes, gel stain for 20 minutes, gel de stain for 45 minutes and Gel Storage for at least one hour.
Finally, our gels are ready for viewing using Gel Doc.
96-well plate

Tray with ice

Pipettor

Vortex

Shaker

SDS Gel Tank

Centrifuge




SDS Gel loaded with proteins

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